Over the past decade a great deal of research has been focused on the development of micro-total-analytical systems. This technology is based on the concept of integration of a series of microfluidic channels for the movement, separation, reaction, and/or detection of various chemicals, e.g., proteins, DNA, chemical compounds, etc.
Prior methods for concentrating analytes include stacking and focusing. In the context of this disclosure, focusing refers to methods for manipulating the velocity of an analyte and thereby causing the analyte to move towards a point at which its velocity is zero and where the analyte will therefore accumulate and increase in concentration. In addition, the location of the zero velocity point is often dependent upon some characteristic of the analyte molecule being focused, so that different analyte molecules are focused at different points, and thereby separated.
In this context, focusing is to be distinguished from stacking, which is a related class of methods in which analytes are moved through a velocity gradient (which is often transient) and the analyte peaks become narrower and more concentrated, but there is no point of zero analyte velocity. In stacking methods, the maximum degree to which analyte concentration can be increased is theoretically limited to the ratio of the velocities on the fast and slow sides of the velocity gradient. In contrast, for focusing at a zero velocity point, there is no theoretical limit to the concentration factor.
Previously known focusing methods include isoelectric focusing (hereinafter “IEF”), electric field gradient focusing (hereinafter “EMF”), counteracting chromatographic electrophoresis, and temperature gradient focusing (hereinafter “TGF”). In general, all of these previously known methods work by creating a gradient in the electrophoretic velocity of the analyte. Therefore, they only work with analytes that have non-zero electrophoretic mobility. For example, in IEF, the analyte has zero electrophoretic mobility only at the zero velocity point.
Micellar electrokinetic chromatography and related methods (hereinafter “EKC”) take advantage of an analyte's affinity for a pseudostationary phase to facilitate separations using capillary electrophoresis. Traditional EKC separations differ from traditional focusing techniques in that analyte molecules move along a separation channel at an essentially constant velocity, whereas in focusing techniques, analytes migrate through the channel to a point where they have a zero velocity. Separation in EKC is achieved because different analytes migrate with different velocities dependent on their affinity for the pseudostationary phase. Often EKC separations are implemented in conjunction with stacking procedures to preconcentrate analytes and facilitate lower detection limits.
In EKC (and in chromatography in general), the buffer is referred to as the mobile phase, and a second phase such as a micellar phase is referred to as the pseudostationary phase. The pseudostationary phase serves the same function—to provide selectivity—as a stationary phase in chromatography, but is not actually stationary, and can move with the buffer and/or with its own electrophoretic mobility.